Shigella species and Salmonella species are major causes of diarrheal disease in the United States. In this project we are investigating two aspects of bacterial virulence which are required for the ability of enteric bacteria to cause disease. A hallmark of bacillary dysentery is the fact that as few as 10 bacteria are sufficient to cause disease. In contrast the infective dose for Salmonella gastritis is greater than 100,000 bacteria whereas over a million Vibrio cholera are required to cause cholera. Because of its extremely low infectious dose, shigellosis is an important cause of diarrheal disease in day care centers, nursing homes and wherever high population density or hygienic conditions favor person to person spread. Under these conditions a single case of bacillary dysentery can spread rapidly through such populations. Aside from shigellosis, the only other bacterial diarrhea associated with such low infectious dose is that caused by strains of Escherichia coli such as the "MacDonald hamburger" strains (E. coli 0157). These strains are among a group of newly emerging infectious agents which have become increasingly important both as diarrheal agents and as a cause of a more serious disease, HUS, or hemolytic uremic syndrome. A major focus of our research is to determine how Shigella and enterohemorrhagic E. coli are able to pass through the stomach with a pH of less than 3.0 in order to reach the intestines. In previous work in our laboratory we had shown that Shigella species are extremely resistant to acid, a trait which is likely to be crucial for the ability of organisms to cause infection at a low infective dose. We have identified a number of genes, rpoS, gadC, and hdeA/B which are required for acid resistance. In recent work we have shown that gadC is part of an operon required for acid resistance and this gene is part of a regulatory cascade in which rpoS places a major role. Data from physiological studies shows that the GadC protein is likely to be involved in the transport of glutamate and glutamine and that the presence of these amino acids is required for the expression of acid resistance. We have developed a model based on this data showing how the GadC operon enables bacteria to survive at low pH. Interestingly, both gadC and hdeA/b have a very restricted distribution among enteric bacteria; they are found only in Shigella species and E. coli. The absence of this operon in either Vibrio or Salmonella species may explain the higher infective dose of these organisms. A particularly important application of these findings, is that the introduction of such genes into Salmonella may facilitate the development of oral vaccines for a number of pathogens. In addition, findings that specific amino acids are required for expression of acid resistance has implications for the field of food microbiology and may lead to a better understanding of how the presence of specific foods in the stomach of infected individuals may augment the likelihood of contracting diarrheal disease. A second area of focus in the laboratory is the area of host specificity. Whereas Shigella species infects only a human host, species of Salmonella vary widely in their host range. For example, S. gallinarum causes severe disease in poultry flocks, but is avirulent in humans. Conversely, S. typhi, the causative agent of typhoid fever is only pathogenic for humans. S. typhimurium, causes a systemic disease in mice similar to typhoid fever, but causes local gastroenteritis in humans. These differences in host specificity suggest that some pathogens have evolved strategies to take advantage of specific host 21 - 12 determinants. The identification of genes encoding host specificity determinants is an exciting area of inquiry which has been little explored. In our lab we are using S. typhimurium and S. gallinarum to study host specificity. Our strategy is to change the host specificity of S. gallinarum by introducing genes from S. typhimurium into S. gallinarum to make a mouse-virulent S. gallinarum. This strategy has begun to yield significant incite into how Salmonella colonize the host and should lead to major findings in the filed of bacterial pathogenesis.